1. Field of the Invention
The present invention relates to simple and reliable methods for negatively charged polymer detection, namely for sequence-selective nucleic acid detection. More specifically, the present invention relates to sequence-selective nucleic acid detection methods, which are essential for the rapid diagnosis of infections and a variety of diseases.
2. Description of the Related Art
Complexes of polythiophene derivatives bearing sulfonic acid moieties and one or several adequately designed amine-containing molecules (electrostatic interactions) have been shown to be responsive to external stimuli (PCT/CA98/01082). More specifically, they were shown to undergo striking conformational changes when exposed to heat, light or various chemical and biochemical moieties giving rise to thermochromism, photochromism, ionochromism or even biochromism. These sulfonic acid-bearing polythiophene derivatives are not positively charged and thus do not have any particular affinity for negatively charged polymers.
The search for methods for sequence-selective nucleic acid detection has evolved into an important research field and has subsequently drawn the attention of researchers from various disciplines such as chemistry, physics, biochemistry, etc. As a result, some interesting DNA hybridization sensors have recently been proposed. (Fodor, S. P. A et al. Science 251, 767, (1991); Livache, T. et al. Nucleic Acids Res. 22, 2912 (1994); Tyagi, S.; Kramer, F. R. Nature Biotechnology 14, 303 (1996); Mikkelson, S. R. Electroanalysis 8, 15 (1996); Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 289, 1757, (2000)).
However, most of these newly developed approaches perform detection by attaching a fluorescent or electro-active tag to the analyte.
Assays that do not require nucleic acid functionalization prior to detection are of greater fidelity and several research groups have reported the utilization of conjugated field-responsive polymers (polypyrroles, polythiophenes, etc.) as electrochemical or optical transducers. (Leclerc, M. Adv. Mater. 11, 1491, (1999); McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 100, 2537 (2000); Chen, L., McBranch, D. W., Wang, H. L., Hegelson, R., Wudl, F. & Whitten, D. G. Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer. PNAS 96, 12287 (1999); Ewbank, P. C., Nuding, G., Suenaga, H., McCullough, R. D., Shinkai, S. Amine functionalized polythiophenes: synthesis and formation of chiral, ordered structures on DNA substrates. Tetrahedron Lett. 42, 155 (2001)). Indeed, the ability of some oligonucleotide-functionalized conjugated polymers to transduce hybridization events into an electrical or optical signal, without utilizing any labeling of the analyte, has been demonstrated. (Youssoufi, H. K.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc., 119, 7388, (1997); Bauerle, P.; Emge, A. Adv. Matter. 10, 324, (1998); Garnier, F.; Youssoufi, H. K.; Srivastava, P.; Mandrand, B.; Delair, T. Synth. Metals, 100, 89, (1999)). The detection mechanism is based on a modification of the electrical and/or optical properties through the capture of the complementary oligonucleotides.
There thus remains a need for simpler, more sensitive and more reliable methods for the rapid and specific identification of nucleic acids. These nucleic acids could be used for the diagnosis of infections and disease. Ideally, an assay that does not require nucleic acid functionalization (chemical manipulation of nucleic acids) prior to detection nor complex reaction mixtures would have the following characteristics: it would be simpler to use than the assays currently available and it would have a high degree of fidelity. Such an assay would be highly beneficial and therefore very desirable.
The present invention seeks to meet these and other needs.